Polypeptide compound and preparation method and use thereof

ABSTRACT

The invention discloses a polypeptide compound and a preparation method and application thereof. The polypeptide compound has a structural formula as follows: (X A X B X C X D X E -X) 2 KY, or {(X A X B X C X D X E -X) 2 K} 2 KY, or {({X A X B X C X D X E -X} 2 K) 2 K} 2 KY, where, X A  is a polar amino acid molecule, X B  and X E  are alkaline amino acid molecules (the same or different), X C  and X D  are non-polar amino acid molecules (the same or different), K is lysine (Lys, K), and X and Y are null, or any one or more amino acid or chemical groups. The polypeptide compound provided in the invention has an effect of enhancing the immune function of a body and has an application potential of being developed into a clinical medicine capable of enhancing the immune function of a body.

FIELD OF THE INVENTION

The present invention relates to the bio-pharmaceutical field, inparticular to a polypeptide compound molecule, a preparation method ofthe polypeptide compound, and an application of the polypeptide compoundin preparation of medicines for enhancing immunity ability and thevaccine immune response ability of an animal body.

BACKGROUND OF THE INVENTION

The genetic genes of organisms are stored in poly deoxynucleotidechains, and proteins that execute biological functions are coded in thegenetic genes. Various proteins exist in organisms and they executedifferent biological functions to maintain vital activities. Thoughthere are numerous kinds of proteins, they are essentially composed of20 kinds of naturally-occurring amino acids that exist in the naturalworld. Proteins differ significantly owing to the composition andsequence of these amino acids. Generally speaking, molecules thatcontain 50 or more amino acids are referred to as proteins, peptidechains that contain 10 or more amino acids are referred to aspolypeptides, and peptide chains that contain less than 10 amino acidsare referred to as oligopeptides. The smallest functional small-peptidediscovered up to now only contains 2 amino acids. Usually, functionalpeptide fragments that are composed of 4 or more amino acids arecommonly seen.

As the Human Genome Project has been completed and the Human ProteomeProject has been developed, more and more functional protein segmentswill be discovered and applied as medicines in the bio-pharmaceuticalfield. A functional protein segment usually refers to a naturalpolypeptide segment that is found as having a specific biologicalfunction. Such a functional protein segment usually is a peptide segmentcomposed of two to ten amino acids. The identified and discoveredfunctional protein segments can be prepared via an artificial synthesisapproach. Polypeptide medicines that have been developed and appliedclinically include “oxytocin”, “thymosin α1”, and “thymopentin”, etc.Polypeptide medicines available presently include “octreotide”, which isprepared through artificial modification of natural peptide chains andused to treat hemorrhage of the digestive tract and acromegaly, and“hirudin peptide”, which has an anti-coagulation effect. The functionalsegments in proteins often can be screened for polypeptide segments thatcontain tens of amino acids or even as few as two amino acids. Thesefunctional segments set a basis for artificial synthesis and applicationof functional polypeptide segments.

In proteins, polypeptides or oligopeptides, the deletion, addition orsubstitution of a single amino acid, the blocking of an amino terminal(N terminal) or carboxyl terminal (C terminal) amino acid, or theaddition of any chemical group into the sequence or at the free end,etc., might result in changes of the original biological activity of theproteins, polypeptides, or oligopeptides. Designing, screening, anddiscovering new functional peptide fragments or seeking for efficientpeptide fragments is an important link in the development of medicines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the purification process.

CONTENTS OF THE INVENTION

To overcome the technical defects in the prior art, in a first aspect,the present invention provides a branched polypeptide compound, whichhas a structural formula expressed as (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY,or {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY, or{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY;

where, X_(A) is a polar amino acid molecule, X_(B) and X_(E) arealkaline amino acid molecules (the same or different), X_(C) and X_(D)are non-polar amino acid molecules (the same or different), K is lysine(Lys, K), and X and Y are null, or one or more amino acid molecules orchemical groups.

The (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY structure is shown in formula 4:

Where, X and Y are null, or any amino acid, or peptide fragmentscomposed of any number of amino acids, or chemical groups that canconnect amino acids or peptide fragments, and X and Y may be the same ordifferent from each other; for example, X may be null, and Y may beglycine (Gly, G).

X_(A) is selected from polar amino acids, and may be cysteine (Cys, C),glycine (Gly, G), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr,Y), asparagine (Asn, N), or glutamine (Gln, Q); preferably is cysteine(Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y),asparagine (Asn, N), or glutamine (Gln, Q); X_(A) more preferably iscysteine (Cys, C), serine (Ser, S), threonine (Thr, T), or glutamine(Gln, Q).

X_(B) and X_(E) are selected from alkaline amino acids, and may bearginine (Arg, R), lysine (Lys, K), or histidine (His, H) respectively,and may be the same or different from each other; X_(B) preferably isarginine (Arg, R) or lysine (Lys, K).

X_(C) and X_(D) are selected from non-polar amino acids, and may bealanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile,I), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), ormethionine (Met, M) respectively, and may be the same or different fromeach other; X_(C) preferably is alanine (Ala, A), valine (Val, V),leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine(Phe, F), or tryptophan (Trp, W), X_(C) more preferably is alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I) or proline (Pro, P); and X_(D)preferably is leucine (Leu, L), isoleucine (Ile, I) or proline (Pro, P).

The present invention further includes a derivative produced throughchemical modification and restructuring of the polypeptide compound, forexample:

A salt compound formed by the polypeptide compound with an organic acidor inorganic acid;

An ether, ester, glucoside, or glycoside compound, etc., which may beformed by the hydroxyl included in the polypeptide compound, but is notlimited to compounds formed in such a way;

A thioether or thioglycoside compound, which may be formed by thesulfhydryl included in the polypeptide compound, or a compoundcontaining disulfide bonds, which may be formed by the sulfhydrylincluded in the polypeptide compound with cysteine or peptide containingcysteine, but is not limited to compounds formed in such a way;

An acylate or alkylate compound, which may be formed by the amido groupincluded in the polypeptide compound, or a glucoside compound, etc.,which may be formed by the amino group included in the polypeptidecompound with saccharides, but is not limited to compounds formed insuch a way;

An ester or amide compound, etc., which may be formed by the carboxylgroup included in the polypeptide compound, but is not limited tocompounds formed in such a way;

A glucoside, acylate, or alkylate compound, etc., which may be formed bythe imino group included in the polypeptide compound, but is not limitedto compounds formed in such a way;

An ester, ether, glucoside, or glycoside compound, which may be formedby the phenolic hydroxyl group included in the polypeptide compound, ora salt compound, which may be formed by the phenolic hydroxyl groupincluded in the polypeptide compound with organic alkali or inorganicalkali compounds, but is not limited to compounds formed in such a way;

A coordinate, clathrate, or chelate compound formed by the polypeptidecompound with metal ions;

A hydrate or solvent formed by the polypeptide compound.

In a second aspect, the present invention provides a pharmaceuticalcomposition that contains the above-mentioned polypeptide compound, ageometrical isomer of the pharmaceutical composition, a pharmaceuticallyacceptable salt or solvated compound of the pharmaceutical composition,and the pharmaceutical composition in a form of pharmaceutical carrieror excipient.

In a third aspect, the present invention provides a method for preparingthe above-mentioned polypeptide compound, in which a synthesis route of(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY is expressed by formula 1:

Y is covalently attached to a WANG solid resin first, and then isconnected with lysine (Lys, K) by condensation;

Next, the two terminal amino groups of K in KY are bonded with twoX_(A)X_(B)X_(C)X_(D)X_(E)-X segments to form a two-branch peptide(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solid resin complex, wherein, theX_(A)X_(B)X_(C)X_(D)X_(E)-X segments may be synthesized first and thencondensed with the WANG solid resin-YK, or X, X_(E), X_(D), X_(C), X_(B)and X_(A) may be bonded in sequence on the basis of the WANG solidresin-YK;

Finally, the two-branch peptide is cleaved from the WANG solid resin, toobtain a polypeptide compound (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with twocopies of the polypeptide X_(A)X_(B)X_(C)X_(D)X_(E)-X.

The synthesis route of {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY structure isexpressed by formula 2:

Y is covalently attached to the WANG solid phase first, and then isconnected with lysine (Lys, K) by condensation, to form a KY-WANG solidresin complex;

Then, the two amino terminals of K in KY are connected with the terminalcarboxyl groups of another two lysines K by condensation, to form atwo-branch skeleton K₂KY-WANG solid resin;

Next, the two amino terminals of each lysine K in “K₂” are connectedwith an X_(A)X_(B)X_(C)X_(D)X_(E)-X segment respectively bycondensation, to form a four-branch peptide{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY-WANG solid resin complex;

Finally, the four-branch peptide is cleaved from the WANG solid resin,to obtain a polypeptide compound {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KYwith four copies of the polypeptide X_(A)X_(B)X_(C)X_(D)X_(E)-X.

The synthesis route of the {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KYstructure is expressed by formula 3:

Y is covalently attached to the WANG solid phase first, and then isconnected with lysine (Lys, K) by condensation, to form KY-WANG solidresin;

Then, the two terminal amino groups of K in KY are bonded with thecarboxyl terminals of another two lysines K by condensation, to form atwo-branch skeleton K₂KY-WANG solid resin complex;

Then, the two terminal amino groups of each K in “K₂” in the K₂KY-WANGsolid resin complex are bonded with the terminal carboxyl groups of theother two lysines K by condensation, to form a four-branch skeletonK₄K₂KY-WANG solid resin complex;

Next, the two terminal amino groups of each lysine K in “K₄” are bondedwith an X_(A)X_(B)X_(C)X_(D)X_(E)-X segment respectively bycondensation, to form an eight-branch peptide{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY-WANG solid resin complex;

Finally, the eight-branch peptide is cleaved from the WANG solid resin,to obtain a polypeptide compound{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY with eight copies of thepolypeptide X_(A)X_(B)X_(C)X_(D)X_(E)-X.

Before the terminal carboxyl group of K is condensed with the Y-WANGsolid resin, the two amido groups of K must be protected, preferablywith at-butyloxycarbonyl (Boc) protection method/group or afluorenylmethoxycarbonyl (Fmoc) protection method/group.

Before the carboxyl terminals of the other two lysines are condensedwith the two amido terminals of K in KY, the two amino groups of eachlysine must be protected; before the carboxyl terminal of theX_(A)X_(B)X_(C)X_(D)X_(E)-X is condensed with the amido terminal of eachlysine, the amino group of the X_(A)X_(B)X_(C)X_(D)X_(E)-X must beprotected, preferably with a t-butyloxycarbonyl (Boc) protectionmethod/group or a fluorenylmethoxycarbonyl (Fmoc) protectionmethod/group.

Specifically, the steps are as follows:

Step 1: protecting the two amino groups of the lysine K with an Fmocprotection method/group;

Step 2: fixing KY to the WANG solid resin with an automatic polypeptidesynthesizer, in the following bonding sequence: WANG solid resin-YK;

When the two-copy polypeptide compound is prepared, the two activatedterminal amino groups of lysine in KY are further condensed with anothertwo X_(A)X_(B)X_(C)X_(D)X_(E)-X fragments, to obtain the polypeptidecompound (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with two copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X, which are connected to the WANG solidresin; or the two activated terminal amino groups of the lysine arefurther condensed with another two lysines K, in each of which the twoamido groups have been protected with an Fmoc protection method, toobtain a two-branch skeleton (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solidresin complex;

When the four-copy polypeptide compound is prepared, the two activatedamino terminals of lysine in the two-branch skeleton “KY-WANG solidresin” are further condensed with two lysine molecules and thenX_(A)X_(B)X_(C)X_(D)X_(E)-X fragments, to obtain the polypeptidecompound {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY with four copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X, which is connected to the WANG solid resin;or the two activated terminal amino groups of each lysine in thetwo-branch skeleton The “KY-WANG solid resin” complex is furthercondensed with another two lysines K, in each of which the two amidogroups have been protected with an Fmoc protection method/group, and oneby one the amino acids condensed to make the X_(A)X_(B)X_(C)X_(D)X_(E)-Xfragment obtain a four-branchskeleton(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₄K₂KY-WANG solid resin complex;

When the eight-copy polypeptide compound is prepared, the two activatedterminal amino groups of each lysine in the four-branch skeleton“K₂KY-WANG solid resin” complex are further condensed with Lysine andX_(A)X_(B)X_(C)X_(D)X_(E)-X fragments, to obtain the polypeptidecompound {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY with eight copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X, which is connected to the WANG solid resin;

Finally, the target polypeptide compound is cleaved from the WANG solidresin with a TFA method, to obtain a crude polypeptide compound product;

Step 3: the crude polypeptide compound product is purified with achromatographic column (model: Daiso 018, 10 μm, 100 Å, 50×250 mm),wherein, the mobile phase A is an aqueous solution that contains 0.05%trifluoroacetic acid and 2% acetonitrile, the mobile phase B is 90%acetonitrile/water, the flow rate is 25 mL/min., and the ultravioletdetection wavelength is 220 nm; the eluting peak solution is collectedand then rough freeze-dried, to obtain a white flocculent polypeptidecompound.

In a fourth aspect, the present invention provides an application of theabove-mentioned polypeptide compound in preparation of immunizationmedicines for humans or animals or medicines for enhancing the immunefunction of humans or animals.

In a fifth aspect, the present invention provides an application of apolypeptide compound prepared with the above-mentioned method inpreparation of immunization medicines for humans or animals or medicinesfor enhancing the immune function of humans or animals.

In a sixth aspect, the present invention provides an application of theabove-mentioned polypeptide compound or a polypeptide compound preparedwith the above-mentioned method in preparation of medicines forinhibiting tumor growth in human or animal bodies.

The tumor is a solid tumor (or residual tumor after medical operation)or a hematological tumor (including leukemia and lymphoma) in a humanbody.

The tumor includes but is not limited to sarcoma, liver cancer, coloncancer, lung cancer, stomach cancer, mammary cancer, and cervicalcancer.

In a seventh aspect, the present invention provides an application ofthe above-mentioned polypeptide compound or a polypeptide compoundprepared with the above-mentioned method in preparation ofanti-infection or anti-virus medicines for humans or animals.

In an eighth aspect, the present invention provides an application ofthe above-mentioned polypeptide compound or a polypeptide compoundprepared with the above-mentioned method in molecular tracers.

In a ninth aspect, the present invention provides an application of theabove-mentioned polypeptide compound or a polypeptide compound preparedwith the above-mentioned method in preparation of medicines for treatingdiseases of humans incurred by vascular proliferation (including, butnot limited to application of medicines for treating maculopathy infundus).

In the polypeptide compounds provided in the present invention, fivemulti-copy amino acid molecules in three kinds (polar amino acidmolecules, alkaline amino acid molecules and non-polar amino acidmolecules) are bonded via covalent bonds to form a branched polypeptidecompound. The polypeptide compound is hopeful to be an effectiveingredient in a variety of medicines, and is applicable to preparationof medicines for preventing and treating many diseases. Especially, thepolypeptide compound will be widely applied in preparation of medicinesfor enhancing immunity ability, and can also be used as a moleculartracer in inhibition of vascularization.

The polypeptide compound described in the present invention isinsensitive to catabolic enzymes in a physiological environment, sinceit has a non-natural molecular structure. Therefore, the effectivehalf-life of the polypeptide compound in organisms can be prolongedeffectively, and thereby the biological effect of the polypeptidecompound can last for longer time in the body.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In the present invention, a branched polypeptide molecule(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY, {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KYor {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY, which contains multiplescopies of X_(A)X_(B)X_(C)X_(D)X_(E)-X, is designed and prepared,wherein, X_(A) is a polar amino acid molecule, and may be cysteine (Cys,C), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), tyrosine(Tyr, Y), asparagine (Asn, N), or glutamine (Gln, Q); X_(B) and X_(E)are alkaline amino acid molecules (the same or different), may be one ortwo of arginine (Arg, R), lysine (Lys, K), or histidine (His, H), andmay be the same or different from each other; X_(C) and X_(D) arenon-polar amino acid molecules (the same or different), may be one ortwo of alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine(Ile, I), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W),and methionine (Met, M), and may be the same or different from eachother; K is lysine Fmoc-Lys(Fmoc)-OH that contain two active aminogroups, X and Y are null, or any amino acid, or peptide fragmentscomposed by any number of amino acids, or chemical groups that can bondup amino acids and peptide fragments. By changing the kinds of X_(A),X_(B), X_(C), X_(D) and X_(E), the polypeptide compound provided in thepresent invention can be adapted to treat a variety of diseases.Especially, the polypeptide compound provided in the present inventioncan enhance humoral immunity and cellular immunity ability of human oranimal bodies. Thus, a medicine for enhancing immune function inclinical application (for humans or animals) can be developed from thepolypeptide compound provided in the present invention.

In 1963, an American scientist R. B. Merrifield invented a solid-phasesynthesis method for extending a peptide chain by fixing the carboxylterminal (C terminal) of amino acids in a target peptide to insolubleresin and controlling the amino terminal (N terminals) of amino acidsbonded to the resin to have a condensation reaction with the carboxylterminal of amino acids to be bonded; that is to say, the amino acidsare condensed one by one starting from the carboxyl terminal (Cterminal) of the polypeptide and extend continuously towards the aminoterminal (N terminal) of the polypeptide fragment. Therefore, when thecondensation reaction of the amino acids is executed, the amido and sidechain groups of the amino acids to be bonded must be protected to avoidreaction of them. At present, commonly used protection methods includet-butyloxycarbonsyl (Boc) protection method and fluorenylmethoxycarbonyl(Fmoc) protection methods/groups. Therefore, whenever an amino acid hasbeen bonded, a deprotection procedure must be executed (i.e., the aminoon the solid-phase carrier is deprotected first, and then has acondensation reaction with the carboxyl of the next target amino acid tobe bonded among amino acids in excessive quantity, to extend the peptidechain). The process is repeated through such steps, i.e., condensation,washing, deprotection, neutralization, washing, and then next cycle ofcondensation (for bonding the target amino acid) is executed, tillrequired length of target peptide chain to be synthesized is reached.After the synthesis is finished, the target polypeptide is cleaved fromthe resin with a TFA method, to obtain a crude product of targetpeptide.

The purification process is as shown in FIG. 1.

Usually, when a linear-chain polypeptide is synthesized, lysine (Lys, K)Fmoc-Lys(Boc)-OH with one active amino is used, and the amino groups onthe side chains are protected by BOC-OH to prevent them fromparticipating in the condensation reaction. Therefore, only one aminogroup in the lysine (Lys, K) can undergo the condensation reaction, andthereby the amino acids are bonded up one by one, and the peptide chainis extended linearly

However, when the branched skeleton described in the present inventionis synthesized, the branch point is a lysine Fmoc-Lys(Fmoc)-OH with twoactive amino groups, and the amino group on the side chain of the lysinealso participates in the condensation reaction. Therefore, when theamino-acid condensation reaction proceeds from the lysine (Lys, K),branch chains will be developed, and a branched skeleton “>KY-WANG solidresin” will be formed. When the next cycle of condensation of lysineFmoc-Lys(Fmoc)-OH is further executed on that basis, a four-branchskeleton “>K₂KY-WANG solid resin” will be formed; next, when thecondensation of Fmoc-Lys(Fmoc)-OH is continued further, an eight-branchskeleton “>K₄K₂KY-WANG solid resin” will be formed.

In the present invention, the lysine (Lys, K, Fmoc-Lys(Fmoc)-OH) withtwo active amino groups in KY is used as a branch point, and two aminoacids for the next step of the operation are bonded by condensation atthe same time. If X_(A)X_(B)X_(C)X_(D)X_(E)-X is bonded, a two-branchpeptide molecule (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with two copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X will be formed. Or, two K(Fmoc-Lys(Fmoc)-OH) may be bonded first to form a two-branch skeleton,and the two amino acids K for the next step of the operation in “K₂”have two active amido groups respectively; thus, a branch point withfour active amino groups is formed for condensation of amino acids(X_(A)X_(B)X_(C)X_(D)X_(E)-X) in the next step. Through suchcondensation in the sequence of X_(A)X_(B)X_(C)X_(D)X_(E)-X, the peptidechain is extended and a four-branch peptide molecule{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY that contains four copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X is formed. Alternatively, two K(Fmoc-Lys(Fmoc)-OH) may be bonded first to form a two-branch skeleton,and then four K (Fmoc-Lys(Fmoc)-OH) may be bonded to form a four-branchskeleton, and the four amino acids K for the next step of operation in“K₄” have two active amino groups respectively; thus, a branch pointwith eight active amino groups is formed for condensation of amino acids(X_(A)X_(B)X_(C)X_(D)X_(E)-X) in the next step. Through suchcondensation in the sequence of X_(A)X_(B)X_(C)X_(D)X_(E)-X, the peptidechain is extended and an eight-branch peptide molecule{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY that contains eight copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X is formed. In that way, a multi-branchpeptide molecule that contains sixteen copies, thirty-two copies, ormore copies of X_(A)X_(B)X_(C)X_(D)X_(E)-X can be formed.

After the synthesis utilizing the WANG solid resin is finished, thepeptide chain can be cleaved from the WANG solid resin with a TFAmethod, so as to obtain a two-branch, four-branch, or eight-branchpeptide molecule, or a peptide molecule with more branches, as describedabove.

Polypeptide synthesis is a conventional technique presently. Please seeChapter 3 “Chemical Synthesis and Purification of Polypeptides” in thebook “Contemporary Theory and Application of Polypeptide Hormones”authored by Shuli Shen and published by Scientific and TechnicalDocumentation Express (in 1998) for the principle and operation ofsynthesis and purification of polypeptides. The synthesis andpreparation of the polypeptide compound in the present invention may beimplemented with the above-mentioned solid phase synthesis method, butis not limited to that method.

Hereunder the present invention will be further detailed in embodiments,but those embodiments should not be understood as constituting anylimitation to the present invention. Any modification or change made bythose skilled in the art to the embodiments in the present inventionaccording to the reveal in this document shall be deemed as falling inthe scope of the present invention.

Embodiment 1: Synthesis of a Copy of a Peptide Fragment

The polypeptide compound in the present invention has the same copy ofthe peptide fragment X_(A)X_(B)X_(C)X_(D)X_(E)-X, regardless of whetherit is in a two-branch, four-branch, or eight-branch structure. In thecopy of the peptide fragment, X_(A) is a polar amino acid molecule, andmay be selected from cysteine (Cys, C), glycine (Gly, G), serine (Ser,S), threonine (Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), orglutamine (Gln, Q); X_(B) and X_(E) are alkaline amino acid molecules(the same or different), and may be selected from one or two of arginine(Arg, R), lysine (Lys, K), and histidine (His, H); X_(C) and X_(D) arenon-polar amino acid molecules (the same or different), and may beselected from one or two of alanine (Ala, A), valine (Val, V), leucine(Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F),tryptophan (Trp, W), and methionine (Met, M); X is null, or any oneamino acid, or a peptide fragment composed of any number of amino acids,or a chemical group that can bond with amino acids and peptidefragments. The possible combinations are shown in Table 1-4, but thepresent invention is not limited to those combinations.

During the synthesis, an amino acid solid-phase synthesis methodprotected by organic chemical Fmoc protection may be used. The specificoperation is as follows:

Step 1: a commercial raw material “X-WANG solid resin” or “XE-WANG solidresin” is selected first, and the amido terminal of X or XE is protectedwith an Fmoc protection method;

Step 2: X_(E) or X_(D) is selected to condense the amino acids one byone and extend the peptide chain, so as to synthesize anX_(A)X_(B)X_(C)X_(D)X_(E)-X copy peptide fragment:

In this embodiment, the synthesis of the copy peptide fragment isimplemented by condensing the amino acids one by one from the carboxylterminal (C) to the amino terminal (N) of the polypeptide and therebyextending the chain on an automatic polypeptide synthesizer (modelABI433A), and then cracking the target polypeptide from the WANG solidresin with a TFA method after the synthesis is finished.

On the automatic polypeptide synthesizer (model ABI433A), X or X_(E) isfixed to the WANG solid resin (the WANG solid resin is a carrier forFmoc protection in the solid phase peptide synthesis) first, and thenamino acids (X_(E), X_(D), X_(C), X_(B), X_(A) or X_(D), X_(C), X_(B),X_(A)) are bonded by condensation. The actual bonding sequence is WANGsolid resin-X-X_(E)X_(D)X_(C)X_(B)X_(A). Thus, a copy peptide fragmentfixed to the WANG solid resin is obtained. Finally, the target copypeptide fragment is cracked from the WANG solid resin with a TFA method.Thus, a crude product of the X_(A)X_(B)X_(C)X_(D)X_(E)-X copy of thepeptide fragment is obtained.

Step 3: purification of the X_(A)X_(B)X_(C)X_(D)X_(E)-X copy of thepeptide fragment

The crude product is purified with a chromatographic column (model:Daiso C18, 10 μm, 100 Å, 50×250 mm), wherein, the mobile phase A in thechromatographic operation is an aqueous solution that contains 0.05%trifluoroacetic acid and 2% acetonitrile, the mobile phase B is 90%acetonitrile/water, the flow rate is 25 mL/min., and the ultravioletdetection wavelength is 220 nm. The eluting peak solution is collectedand then freeze-dried. Thus, white flocculentX_(A)X_(B)X_(C)X_(D)X_(E)-X copy peptide fragments are obtained.

Then, the copy peptide fragments are packed in a sealed state and storedin a refrigerator for later use; the purity of the copy peptidefragments may be >99%.

X_(A), X_(B), X_(C), X_(D) and X_(E) are commercially available. Whenthe polypeptide compound in the present invention is prepared, the WANGsolid resin-X or WANG solid resin-X_(E) that is purchased commerciallymay be also used as a raw material, and amino acids are condensed to theterminal end of X or X_(E), so as to obtain the copy peptide fragmentdescribed in the present invention. Some of the copy peptide fragmentsobtained through synthesis and their molecular weights measured by massspectrometer measurement are listed in Table 1.

TABLE 1 List of Groups in the Copy Peptide FragmentX_(A)X_(B)X_(C)X_(D)X_(E)-X Molecular Weight Embodiment X_(A) X_(B)X_(C) X_(D) X_(E) X Theoretical Actual 1-1 T K L P R Null 613.75 613.981-2 S R P L R G 684.79 684.93 1-3 C R L P R G 700.86 701.25 1-4 T R P LR C 744.91 745.18 1-5 S K L L K G 644.80 645.32 1-6 Q K A P H Null579.65 580.04 1-7 Y R F M K A 815.01 815.53 1-8 C K L P R Null 615.79616.24

It is seen from the result in Table 1: the deviation of the measuredmolecular weight of the copy peptide fragment synthesized in the presentinvention from the theoretical molecular weight is less than 1%, whichproves that the copy of the peptide fragment is the correct copy of thepeptide fragment in the corresponding embodiment.

This embodiment part is provided to disclose the content of the copypeptide fragment, rather than limit the present invention. The actualsynthesis may be executed according to the description in the followingembodiments.

A branched skeleton is prepared with the method described in the presentinvention, wherein, the amino acid at the branch node isFmoc-Lys(Fmoc)-OH that carries two active amino groups, which provide abranch loci for the subsequent amino-acid condensation and bondingreactions. Therefore, a two-branch skeleton “>KY-WANG solid resin”,four-branch skeleton “>K₂KY-WANG solid resin”, or eight-branch skeleton“>K₄K₂KY-WANG solid resin”, . . . , can be synthesized. Then, aminoacids in which the amino groups are protected are selected according tothe conventional peptide extension reaction, and peptide extension fromthe branch nodes is executed, so as to prepare the two-branch peptidemolecule (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY, four-branch peptide molecule{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY, or eight-branch peptide molecule{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₁₂KY of the present invention. Inview of the peptide chain extension process being a mature technique,the details of the synthesis steps will not be described any furtherhere.

Embodiment 2: Synthesis of a Two-Branch Peptide Molecule(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY

The structure and synthesis route of the polypeptide compound(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with two copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X provided in the present invention are shownin formula 1, wherein, X_(A) is a polar amino acid molecule, X_(B) andX_(E) are alkaline amino acid molecules, X_(C) and X_(D) are non-polaramino acid molecules, K is lysine Fmoc-Lys(Fmoc)-OH that contains twoactive amino groups, and X and Y are null, or any one or more aminoacids or chemical groups:

In this embodiment, an amino acid solid-phase synthesis method protectedby an organic chemical Fmoc protection method is used. The specificoperation is as follows:

Step 1: The amido groups of the lysine are protected with an Fmocprotection method;

Step 2: The (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY polypeptide compound issynthesized:

In this embodiment, the synthesis of the polypeptide is implemented bycondensing the amino acids one by one from the carboxyl terminal (C) tothe amino terminal (N) of the polypeptide and thereby extending thechain on an automatic polypeptide synthesizer (model ABI433A), and thencracking the target polypeptide from the WANG solid resin with a TFAmethod after the synthesis is finished.

On the automatic polypeptide synthesizer (model ABI433A), Y is fixed tothe WANG solid resin (the WANG solid resin is a carrier for Fmocprotection in the solid phase peptide synthesis) first, and then lysine(Fmoc-Lys(Fmoc)-OH) is bonded by condensation. The actual bondingsequence is WANG solid resin-Y-Lys. Thus, a two-branch skeleton“>KY-WANG solid resin” with branch nodes is formed; since the terminallysine has two activated amino groups, the two active amino terminals ofK in the “>KY-WANG solid resin” will react with another twoX_(A)X_(B)X_(C)X_(D)X_(E)-X segments. Thus, an extended two-branchpeptide (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂Y-WANG solid resin is obtained,i.e., a polypeptide compound (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solidresin with two copies of X_(A)X_(B)X_(C)X_(D)X_(E)-X, which are fixed tothe WANG solid resin, is obtained.

In this step, X_(A)X_(B)X_(C)X_(D)X_(E)-X segments may be synthesized asdescribed in embodiment 1 first, and then they may be condensed with theWANG solid resin-Y-Lys; or, the two active amino terminals of K in the“>KY-WANG solid resin” may be bonded with X, X_(E), X_(D), X_(C), X_(B)and X_(A) in sequence. Finally, the target polypeptide compound may becracked from the WANG solid resin with a TFA method, to obtain a crudeproduct of a (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY polypeptide compound.

Step 3: purification of (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY polypeptidecompound

The crude product is purified using a chromatographic column (model:Daiso C18, 10 μm, 100 Å, 50×250 mm), wherein, the mobile phase A in thechromatographic operation is an aqueous solution that contains 0.05%trifluoroacetic acid and 2% acetonitrile, the mobile phase B is 90%acetonitrile/water, the flow rate is 25 mL/min., and the ultravioletdetection wavelength is 220 nm. The eluting peak solution is collectedand then freeze-dried. Thus, a white flocculent(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY polypeptide compound is obtained. Then,the polypeptide compound is packed in a sealed state and stored in arefrigerator for later use; the purity of the polypeptide compound maybe >99%.

X_(A), X_(B), X_(C), X_(D) and X_(E) are commercially available. Whenthe polypeptide compound in the present invention is prepared, the WANGsolid resin-Y that is purchased commercially may also be used as a rawmaterial, and the amino acids may be condensed to the terminal portionsof Y with the above-mentioned method, so as to obtain the polypeptidecompound in the present invention.

A series of two-branch peptide molecules(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY are obtained with the method describedin this embodiment, and are shown in Table 2.

TABLE 2 List of Groups in the Two-Branch Peptide Molecule(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY Embodiment X_(A) X_(B) X_(C) X_(D)X_(E) X Y 2-1 T K L P R Null G 2-2 S R P L R G A 2-3 C R L P R G G 2-4 TR P L R C G 2-5 S K L L K G Null 2-6 Q K A P H Null G 2-7 Y R F M K A C2-8 C K W A H Null A 2-9 S K L I K Null G 2-10 C R L A K A Null 2-11 S RL F R G W 2-12 T K P P R Null G 2-13 T R P L K Null G 2-14 S K I L RNull G 2-15 C H V M K P I 2-16 N H L W H N DEmbodiment 3: Synthesis of a Four-Branch Peptide Molecule{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY

The structure and synthesis route of the polypeptide compound{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY with four copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X as provided in the present invention areshown in formula 2.

The structure may also be expressed as(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₄K₂KY. The definitions of the groups arethe same as those in the embodiment 2:

In this embodiment, an amino acid solid-phase synthesis method protectedby an organic chemical Fmoc protection method is used. The specificoperation is as follows:

Step 1: The amido groups of the lysine are protected with an Fmocprotection method;

Step 2: The {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY polypeptide compound issynthesized:

In this embodiment, the synthesis of the polypeptide is implemented bycondensing the amino acids one by one from the carboxyl terminal (C) tothe amino terminal (N) of the polypeptide and thereby extending thechain on an automatic polypeptide synthesizer (model ABI433A), and thencracking the target polypeptide from the WANG solid resin with a TFAmethod after the synthesis is finished.

On the automatic polypeptide synthesizer (model ABI433A), Y is fixed tothe WANG solid resin (the WANG solid resin is a carrier for Fmocprotection in the solid phase peptide synthesis) first, and then lysine(Fmoc-Lys(Fmoc)-OH) is bonded by condensation. The actual bondingsequence is WANG solid resin-Y-Lys. Thus, a two-branch skeleton“>KY-WANG solid resin” with branch nodes is formed; since the terminallysine has two activated amino groups, the two activated amino groupswill undergo a condensation reaction with the carboxyl terminals ofanother two lysines (K, here, the two amino groups are protected). Thus,two extended branch skeletons of the K₂KY-WANG solid resin are obtained.Here, the amino terminals of the lysine in KY are bonded with twolysines (K), each of which has two active amino groups; thus, afour-branch skeleton “>K₂KY-WANG solid resin” with branch nodes isformed; the condensation with two X_(A)X_(B)X_(C)X_(D)X_(E)-X segmentsis executed further on each lysine (K) that has two active amino groupsin “K₂”; thus, a polypeptide compound{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY-WANG solid resin with four copiesof X_(A)X_(B)X_(C)X_(D)X_(E)-X, which is fixed to the WANG solid resin,is obtained.

In this step, X_(A)X_(B)X_(C)X_(D)X_(E)-X segments may be synthesized asdescribed in embodiment 1, and then they may be condensed with theK₂KY-WANG solid resin; or, the two active amino terminals of K in the“>K₂KY-WANG solid resin” may be bonded with X, X_(E), X_(D), X_(D),X_(B) and X_(A) in sequence. Finally, the target polypeptide compoundmay be cracked from the WANG solid resin with a TFA method, to obtain acrude product of {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY polypeptidecompound.

Step 3: purification of {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY polypeptidecompound

The purification is the same as that in the embodiment 2. Finally, awhite flocculent {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY polypeptidecompound is obtained. Then, the polypeptide compound is packed in asealed state and stored in a refrigerator for later use; the purity ofthe polypeptide compound may be >99%.

A series of four-branch peptide molecules{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY are obtained using the methoddescribed in this embodiment. Please see Table 3 for the selection ofthe groups.

TABLE 3 List of Groups in the Four-Branch Peptide Molecule{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY Embodiment X_(A) X_(B) X_(C) X_(D)X_(E) X Y 4-1 T K L P R Null G 4-2 S R P L R G A 4-3 C R L P R G G 4-4 TR P L R Null G 4-5 S K L L K G Null 4-6 Q K A P H Null G 4-7 Y R F M K AC 4-8 C K W A H Null A 4-9 S K L I K Null G 4-10 C R L A K A Null 4-11 SR L F R G W 4-12 T K P P R Null G 4-13 T R P L K Null G 4-14 S K I L RNull G 4-15 C H V M K P I 4-16 N H L W H N DEmbodiment 4: Synthesis of Eight-Branch Peptide Molecule{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}2K)₂K}₂KY

The structure and synthesis route of the polypeptide compound{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}2K)₂K}₁₂KY with eight copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X provided in the present invention are shownin formula 3. The structure may also expressed as(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₈K₄K₂KY. The definitions of the groups arethe same as those in embodiment 2:

In this embodiment, an amino acid solid-phase synthesis method protectedby an organic chemical Fmoc protection method is used. The specificoperation is as follows:

Step 1: The amino groups of the lysine are protected with an Fmocprotection method;

Step 2: synthesis of {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}2K)₂K}₂KYpolypeptide compound:

In this embodiment, the synthesis of the polypeptide is implemented bycondensing the amino acids one by one from the carboxyl terminal (C) tothe amino terminal (N) of the polypeptide and thereby extending thechain on an automatic polypeptide synthesizer (model ABI433A), and thencracking the target polypeptide from the WANG solid resin with a TFAmethod after the synthesis is finished.

On the automatic polypeptide synthesizer (model ABI433A), Y is fixed tothe WANG solid resin (the WANG solid resin is a carrier for Fmocprotection in the solid phase peptide synthesis) first, and then lysine(Lys, K) is bonded by condensation. The actual bonding sequence is WANGsolid resin-Y-Lys. Thus, a two-branch skeleton “>KY-WANG solid resin”with branch nodes is formed; since the terminal lysine has two activatedamino groups, the two activated amino groups will have a condensationreaction with the carboxyl terminals of another two lysines (K, here,the two amino groups are protected). Thus, two extended branch skeletonsK₂KY-WANG solid resin are obtained. Here, the amino terminals of thelysine in KY are bonded with two lysines (K, here, the two amino groupsare protected), each of which has two active amino groups; thus, afour-branch skeleton “>K₂KY-WANG solid resin” with branch nodes isformed; the terminal ends of two lysines that have a single activatedamino group each in “K₂” have a condensation reaction with the carboxylterminals of another two lysines (K, here, the two amino groups areprotected). Thus, extended four-branch skeletons of the K₄K₂KY-WANGsolid resin are obtained. Here, the amino terminals of the terminallysine in K₂KY are bonded with four lysines (K), each of which has twoactive amino groups; thus, an eight-branch skeleton “>K₄K₂KY-WANG solidresin” with branch nodes is formed; the condensation with twoX_(A)X_(B)X_(C)X_(D)X_(E)-X segments is executed further on each lysine(K) that has two active amino groups in “K₄” in the “K₄K₂KY-WANG solidresin”; thus, a polypeptide compound{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY-WANG solid resin with eightcopies of X_(A)X_(B)X_(C)X_(D)X_(E)-X, which is fixed to the WANG solidresin, is obtained.

In this step, the X_(A)X_(B)X_(C)X_(D)X_(E)-X segments may besynthesized first, and then they may be condensed with the{(K)₂K}₂KY-WANG solid resin; or, the two active amino terminals of K inthe “>K₄K₂KY-WANG solid resin” may be bonded with X, X_(E), X_(D),X_(C), X_(B) and X_(A) in sequence. Finally, the target polypeptidecompound may be cracked from the WANG solid resin with a TFA method, toobtain a crude product of {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KYpolypeptide compound.

Step 3: purification of {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KYpolypeptide compound

The purification is the same as that in embodiment 2. Finally, a whiteflocculent {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY polypeptide compoundis obtained. Then, the polypeptide compound is packed in a sealed stateand stored in a refrigerator for later use; the purity of thepolypeptide compound may be >99%.

A series of eight-branch peptide molecules{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY are obtained with the methoddescribed in this embodiment. Please see Table 4 for the selection ofthe groups.

TABLE 4 List of Groups in the Eight-Branch Peptide Molecule{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY Embodiment X_(A) X_(B) X_(C)X_(D) X_(E) X Y 8-1 T K L P R Null G 8-2 S R P L R G A 8-3 C R L P R G G8-4 T R P L R Null G 8-5 S K L L K G Null 8-6 Q K A P H Null G 8-7 Y R FM K A C 8-8 C K W A H Null A 8-9 S K L I K Null G 8-10 C R L A K A Null8-11 S R L F R G W 8-12 T K P P R Null G 8-13 T R P L K Null G 8-14 S KI L R Null G 8-15 C H V M K P I 8-16 N H L W H N D

Since the polypeptide compound in the present invention is a type oforganic molecule with biological activity, their biological effectsdepend on their amino acid sequence and structure. Any change of asingle amino acid in the protein or peptide sequence may result inchanges of the biological activity. Hereunder the biological activityand efficacy of the polypeptide compound provided in the presentinvention will be described in specific experimental examples.

EXPERIMENTAL EXAMPLE 1 Experiment of the Immune Effect of thePolypeptide Compound Provided in the Present Invention Among Birds andPoultry (Chicks)

Newcastle Disease Virus (NDV) can cause a hemagglutination phenomenonamong chickens, which is a specific antibody neutralization reaction.The principle is that the hemagglutinin produced by the virus can causeagglutination of red blood cells.

However, if a specific antibody is used to counteract the virus firstbefore the virus is added into red blood cells, the hemagglutinationphenomenon will not occur any more. Such a test is referred to as aHemagglutination inhibition test (HI), and the maximum multiple ofdilution of the anti-serum used in the detection is the titer of theantibody. The higher the titer of the tested antibody, the better theimmune effect is.

The HI method has the following advantages:

1. High sensitivity: the HI method can detect antibody in a tracequantity, and the result is relatively accurate, the reaction is one ofsensitive serologic detection reactions;

2. High specificity: the virus that causes agglutination of red bloodcells can only be inhibited by a specific antibody;

3. High detection speed: only about 2h is required in a HI test to judgethe result;

4. The HI test doesn't have any high requirement for the environment,and the operation is simple and quick, a large quantity of samples canbe detected in one test.

The polypeptide compounds obtained in the embodiments 2-1≠2-16,4-1≠4-16, and 8-1≠8-16 in the present invention are used for testing ofthe antibody titer among chicks: Live NDV vaccine (CS2 strain, fromChengdu Tecbond Biological Products Co., Ltd.) and the polypeptidecompound provided in the present invention are inoculated into SPFchicks, and then the HI antibody formation effect of the polypeptidecompound against live NDV vaccine in the bodies of SPF chicks is tested,so as to ascertain the immune effect of the polypeptide compoundprovided in the present invention against live NDV vaccine (antigen).

The experimental method is as follows: 7-day-old specific pathogen freechicks (abbreviated as SPF chicks) are chosen. The SPF chicks aredivided into 8 groups, with 12 chicks in each group. Subcutaneousvaccination is carried out in the axillary region of a wing of each SPFchick in the following groups. The SPF chicks in each group are bred inisolators. About 1 ml venous blood is taken under a wing of each SPFchick on the fourteenth day after inoculation, the serum is separated,and the HI detection is carried out. The detection results of theembodiments 2-5, 2-6, 4-9, 4-12, 8-13 and 8-14 (corresponding to theexperimental groups 1≠6 sequentially) are shown in Table 5 (only a partof the detection results of the polypeptide compounds are listed). Theresults of the other embodiments have little difference with those shownin Table 5, and are omitted here. See the “Experiment Course of AnimalImmunology” authored by Xin Guo and published by the Press of ChinaAgricultural University in 2007 for the details of operation.

Blank group: 0.3 ml normal saline is injected;

Reference group: 0.3 ml live NDV vaccine (abbreviated as “vaccine”, CS2strain) is inoculated;

Experimental group: 0.3 ml vaccine mixed with 0.2 μg polypeptidecompound provided in the present invention is inoculated.

TABLE 5 Result of Immunity Experiment of SPF Chicks Average InoculatedSubstance Antibody Group Vaccine Embodiment Titer Blank group None NoneNegative Reference Vaccine None  8.3log2 group Experimental Vaccine 2-5 9.3log2 group 1 Experimental Vaccine 2-6  9.4log2 group 2 ExperimentalVaccine 4-9  9.7log2 group 3 Experimental Vaccine 4-12  9.8log2 group 4Experimental Vaccine 8-13 10.0log2 group 5 Experimental Vaccine 8-1410.2log2 group 6 Note: “Negative” refers to that the HI antibody titeris zero;

The dietetic activities of the SPF chicks in the groups are normalduring the experiment, no adverse reaction is seen, and no SPF chickdies. That indicates the polypeptide compound provided in the presentinvention is safe to use. The results in Table 5 indicate that theaverage HI antibody titer (experimental groups 1-8) is higher after thepolypeptide compound provided in the present invention is added, whencompared with the blank group and reference group (vaccine is inoculatedsolely). Thus, it is proved that a good immunity enhancement effect canbe attained when the polypeptide compound provided by the presentinvention is used in combination with the vaccine, and the average HIantibody titer is higher than that of the reference by 1 or more.

EXPERIMENTAL EXAMPLE 2 Experiment on the Influence of Different Branchesof the Polypeptide Compound Provided in the Present Invention on theImmune Effect Under a Condition of the Same Multi-Copy Group

An experiment on the immune effect of two groups of polypeptidecompounds (groups I and II) in the present invention is carried out withthe method described in the experimental example 1. In the two groups ofpolypeptide compounds, the multi-copy groups of the polypeptidecompounds in the same group are the same, only the quantities ofbranches are different. The two groups of compounds are selectedrandomly from the embodiments. The detection results of the embodiments2-1, 2-3, 4-1, 4-3, 8-1 and 8-3 are shown in Table 6. The results of theother embodiments have little difference from those shown in Table 5,and are omitted here.

Group division:

Blank group: 0.3 ml normal saline is injected;

Reference group: 0.3 ml live NDV vaccine (abbreviated as “vaccine”, CS2strain) is inoculated;

Experimental group: in the embodiments, 0.3 ml vaccine mixed with 0.2 μgpolypeptide compounds is inoculated;

TABLE 6 Result of Immunity Experiment of SPF Chicks Average InoculatedSubstance Antibody Group Vaccine Embodiment Titer Blank group None NoneNegative Reference group Vaccine None  8.3log2 Experimental Group IVaccine 2-1  9.4log2 group Vaccine 4-1  9.7log2 Vaccine 8-1 10.2log2Group II Vaccine 2-3  9.5log2 Vaccine 4-3  9.9log2 Vaccine 8-3 10.3log2Note: “Negative” refers to that the HI antibody titer is zero.

The dietetic activities of the SPF chicks in the groups are normalduring the experiment. No adverse reaction is seen, and no SPF chickdies. That indicates the polypeptide compound provided in the presentinvention is safe to use.

The results in Table 6 indicates that the immune response effect to thechicks is improved and the immune enhancement effect is further improvedas the number of copies of the polypeptide segment is increased (i.e.,the quantity of branches is increased). That means the biological effectis positively correlated to the number of copies of the peptidefragments.

INDUSTRIAL APPLICABILITY

The polypeptide compounds provided in the present invention are usefuleffective ingredients in a variety of medicines, and are applicable tomedicines for preventing and treating many diseases. Especially, thepolypeptide compounds can be used to prepare medicines for enhancingimmune ability, and are suitable for industrial application.

1-32. (canceled)
 33. A polypeptide compound, having a structural formulaexpressed as (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY or{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY or{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY; wherein, X_(A) is a polaramino acid molecule, X_(B) and X_(E) are alkaline amino acid molecules(the same or different), X_(C) and X_(D) are non-polar amino acidmolecules (the same or different), K is lysine (Lys, K), and X and Y arenull, or one or more of amino acid or chemical groups.
 34. Thepolypeptide compound according to claim 33, wherein, the(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY structure is shown in formula 4:


35. The polypeptide compound according to claim 33, wherein, the{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY structure is shown in formula 5:


36. The polypeptide compound according to claim 33, wherein, the{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY structure is shown in formula6:


37. The polypeptide compound according to claim 33, wherein, X and Y arenull, or any amino acid, or peptide fragments composed of any number ofamino acids, or chemical groups that can connect amino acids or peptidefragments, and X and Y may be the same or different from each other; forexample, X may be null, and Y may be glycine (Gly, G).
 38. Thepolypeptide compound according to claim 33, wherein, X_(A) is selectedfrom cysteine (Cys, C), glycine (Gly, G), serine (Ser, S), threonine(Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), or glutamine (Gln, Q);or X_(A) is selected from cysteine (Cys, C), serine (Ser, S), threonine(Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), or glutamine (Gln, Q);or X_(A) is selected from cysteine (Cys, C), serine (Ser, S), threonine(Thr, T), or glutamine (Gln, Q).
 39. The polypeptide compound accordingto claim 33, wherein, X_(B) and X_(E) are selected from arginine (Arg,R), lysine (Lys, K), or histidine (His, H) respectively, and may be thesame or different from each other; or X_(B) is selected from arginine(Arg, R) or lysine (Lys, K).
 40. The polypeptide compound according toclaim 33, wherein, X_(C) and X_(D) are selected from alanine (Ala, A),valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro,P), phenylalanine (Phe, F), tryptophan (Trp, W), or methionine (Met, M)respectively, and may be the same or different from each other; or X_(C)is selected from alanine (Ala, A), valine (Val, V), leucine (Leu, L),isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F), ortryptophan (Trp, W), more preferably is alanine (Ala, A), leucine (Leu,L), isoleucine (Ile, I) or proline (Pro, P); and X_(D) is selected fromleucine (Leu, L), isoleucine (Ile, I) or proline (Pro, P).
 41. Thepolypeptide compound according to claim 33, further comprising a saltcompound formed by the polypeptide compound with an organic acid orinorganic acid.
 42. The polypeptide compound according to claim 33,further comprising an ether, ester, glucoside, or glycoside compound,formed by the hydroxyl group included in the polypeptide compound. 43.The polypeptide compound according to claim 33, further comprising athioether or thioglycoside compound, formed by the sulfhydryl groupincluded in the polypeptide compound, or further comprising a compoundcontaining disulfide bonds, which may be formed by the sulfhydryl groupsincluded in the polypeptide compound with cysteine or a peptidecontaining cysteine.
 44. The polypeptide compound according to claim 33,further comprising an acylate or alkylate compound, formed by the aminogroups included in the polypeptide compound, or further comprising aglucoside compound formed by the amino group included in the polypeptidecompound with saccharides.
 45. The polypeptide compound according toclaim 33, further comprising an ester or amide compound formed by acarboxyl group included in the polypeptide compound.
 46. The polypeptidecompound according to claim 33, further comprising a glucoside, acylate,or alkylate compound formed by an imino group included in thepolypeptide compound.
 47. The polypeptide compound according to claim33, further comprising an ester, ether, glucoside, or glycoside compoundformed by a phenolic hydroxyl group included in the polypeptidecompound, or further comprising a salt compound formed by a phenolichydroxyl group included in the polypeptide compound with organic alkalior inorganic alkali compounds.
 48. The polypeptide compound according toclaim 33, further comprising a coordinate, clathrate, or chelatecompound formed by the polypeptide compound with metal ions.
 49. Thepolypeptide compound according to claim 33, further comprising a hydrateor solvent formed by the polypeptide compound.
 50. A pharmaceuticalcomposition comprising the polypeptide compound according to claim 33,or a geometrical isomer of the polypeptide compound, a pharmaceuticallyacceptable salt or solvated compound of the polypeptide compound, and apharmaceutical carrier or excipient.
 51. A method for preparing thepolypeptide compound according to claim 33, wherein, a synthesis routeof (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY is expressed by formula 1:

Y is first fixed to a WANG solid resin, and then is bonded with lysineFmoc-Lys(Fmoc)-OH(Lys, K) by condensation, to form a two-branch skeleton“>KY-WANG solid resin” with branch nodes; next, the two active terminalamino groups of K in the “>KY-WANG solid resin” are bonded with aX_(A)X_(B)X_(C)X_(D)X_(E)-X segment respectively, to form a two-branchpeptide (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solid resin; or the twoactive terminal amino groups of K in the “>KY-WANG solid resin” arebonded with amino acids X, X_(E), X_(D), X_(C), X_(B), and X_(A) bycondensation in sequence, to obtain a(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solid resin; Finally, thetwo-branch peptide is cracked from the WANG solid resin and thenpurified, to obtain a polypeptide compound(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with two copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X.
 52. A method for preparing the polypeptidecompound according to claim 33, wherein, a synthesis route of the{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY structure is expressed by formula2:

Y is fixed to WANG solid resin first, and then is bonded with lysineFmoc-Lys(Fmoc)-OH by condensation, to form a two-branch skeleton“>KY-WANG solid resin” with branch nodes; then, the two active terminalamino groups of K in the “>KY-WANG solid resin” are bonded with theterminal carboxyl groups of lysine Fmoc-Lys(Fmoc)-OH by condensation, toform a four-branch skeleton “>K₂KY-WANG solid resin” with two branchnodes; next, the two active terminal amino groups of each lysine K inthe “>K₂KY-WANG solid resin” are bonded with aX_(A)X_(B)X_(C)X_(D)X_(E)-X segment respectively, to form a four-branchpeptide (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY-WANG solid resin; or the twoactive terminal amino groups of K in the “>K₂KY-WANG solid resin” arebonded with amino acids X, X_(E), X_(D), X_(C), X_(B), and X_(A) bycondensation in sequence, to obtain a(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₄K₂KY-WANG solid resin; finally, thefour-branch peptide is cracked from the WANG solid resin and ispurified, to obtain a polypeptide compound{(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY with four copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X.
 53. A method for preparing the polypeptidecompound according to claim 33, wherein, a synthesis route of the{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY structure is expressed byformula 3:

Y is fixed to WANG solid resin first, and then is bonded with lysineFmoc-Lys(Fmoc)-OH by condensation, to form a two-branch skeleton“>KY-WANG solid resin” with branch nodes; the two active terminal aminogroups of K are bonded with the carboxyl terminals of lysineFmoc-Lys(Fmoc)-OH by condensation, to form a four-branch skeleton“K₂KY-WANG solid resin”; then, the two active terminal amino groups of Kin the four-branch skeleton “>K₂KY-WANG solid resin” are bonded with theterminal carboxyl groups of lysine Fmoc-Lys(Fmoc)-OH by condensation, toform an eight-branch skeleton “>K₄K₂KY-WANG solid resin” with fourbranch nodes; next, the two active terminal amino groups of each lysineK in the “>K₄K₂KY-WANG solid resin” are bonded with aX_(A)X_(B)X_(C)X_(D)X_(E)-X segment respectively, to form eight-branchpeptide (X_(A)X_(B)X_(C)X_(D)X_(E)-X)₈K₄K₂KY-WANG solid resin; or thetwo active terminal amino groups of K in the “>K₄K₂KY-WANG solid resin”are bonded with amino acids X, X_(E), X_(D), X_(C), X_(B), and X_(A) bycondensation in sequence, to obtain(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₈K₄K₂KY-WANG solid resin; finally, theeight-branch peptide is cracked from the WANG solid resin and purified,to obtain a polypeptide compound{({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY with eight copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X.
 54. The method according to claim 51,wherein, before the carboxyl terminal of K is condensed with Y-WANGsolid resin, the two amino groups of K are protected with thet-butyloxycarboryl (Boc) protection method or with thefluorenylmethoxycarbonyl (Fmoc) protection method.
 55. The methodaccording to claim 51, wherein, before the carboxyl terminals of theother two lysines are condensed with the two terminal amido groups of Kin KY, the two amido groups of each lysine are protected; before thecarboxyl terminal of the X_(A)X_(B)X_(C)X_(D)X_(E)-X is condensed withthe terminal amido group of each lysine, the amino group of theX_(A)X_(B)X_(C)X_(D)X_(E)-X is protected with the t-butyloxycarboryl(Boc) protection method or with the fluorenylmethoxycarbonyl (Fmoc)protection method.
 56. The method according to claim 51, comprising thefollowing steps: step 1: protecting the two amino groups of the lysine Kwith an Fmoc protection method; step 2: fixing KY to the WANG solidresin with an automatic polypeptide synthesizer, in the followingbonding sequence: WANG solid resin-YK; when the two-copy polypeptidecompound is prepared, the two activated terminal amino groups of lysinein KY are further condensed with another two X_(A)X_(B)X_(C)X_(D)X_(E)-Xfragments, to obtain the polypeptide compound(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂KY with two copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X, which is fixed to the WANG solid resin; orthe two activated terminal amino groups of the lysine in KY are furthercondensed with another two lysines K, in each of which the two amidogroups have been protected with an Fmoc protection method, to obtain atwo-branch skeleton K₂KY-WANG solid resin; when the four-copypolypeptide compound is prepared, the two activated terminal aminogroups of each lysine in the two-branch skeleton “K₂” are furthercondensed with two X_(A)X_(B)X_(C)X_(D)X_(E)-X fragments, to obtain thepolypeptide compound {(X_(A)X_(B)X_(C)X_(D)X_(E)-X)₂K}₂KY with fourcopies of X_(A)X_(B)X_(C)X_(D)X_(E)-X, which is fixed to the WANG solidresin; or the two activated terminal amino groups of each lysine in thetwo-branch skeleton “K₂” are further condensed with another two lysinesK, in each of which the two amino groups have been protected with anFmoc protection method, to obtain a four-branch skeleton K₄K₂KY-WANGsolid resin; or when the eight-copy polypeptide compound is prepared,the two activated terminal amino groups of each lysine in thefour-branch skeleton “K₄” are further condensed with twoX_(A)X_(B)X_(C)X_(D)X_(E)-X fragments, to obtain the polypeptidecompound {({X_(A)X_(B)X_(C)X_(D)X_(E)-X}₂K)₂K}₂KY with eight copies ofX_(A)X_(B)X_(C)X_(D)X_(E)-X, which is fixed to the WANG solid resin;where, the polypeptide compound is cracked from the WANG solid resinwith a TFA method, to obtain a crude polypeptide compound product; step3: the crude polypeptide compound product is purified with achromatographic column (model: Daiso C18, 10 μm, 100 Å, 50×250 mm),wherein, the mobile phase A is an aqueous solution that contains 0.05%trifluoroacetic acid and 2% acetonitrile, the mobile phase B is 90%acetonitrile/water, the flow rate is 25 mL/min., and the ultravioletdetection wavelength is 220 nm; the eluting peak solution is collectedand then freeze-dried, to obtain a white flocculent polypeptidecompound.
 57. A method for enhancing the immune function of humans oranimals comprising administering a compound according to claim
 33. 58. Amethod for enhancing the immune function of humans or animals comprisingadministering a compound prepared according to claim
 51. 59. A methodfor inhibiting tumor growth in humans or animals comprisingadministering a compound according to claim
 33. 60. The method accordingto claim 59, wherein, the tumor is a solid tumor, a residual tumor aftermedical operation, or a hematological tumor, wherein the hematoloigicaltumor is selected from leukaemia and lymphoma, in a human body.
 61. Themethod according to claim 59, wherein, the tumor is selected fromsarcoma, liver cancer, colon cancer, lung cancer, stomach cancer,mammary cancer, and cervical cancer.
 62. An anti-infection or anti-virusmedicine for humans or animals comprising a polypeptide compoundaccording to claim
 33. 63. A molecular tracer comprising a polypeptidecompound according to claim
 33. 64. A medicine for treating diseases ofhumans incurred by vascular proliferation comprising a polypeptidecompound according to claim 33.